The present invention relates to a medicament. More specifically, the present invention relates to a polymer compound comprising a saccharide-conjugated sphingosine, and a medicament comprising the polymer compound as an active ingredient.
Vaccines are generally used for the prevention of viral infections. However, vaccines fail to achieve sufficient preventive effects against viruses which rapidly develop variations, such as influenza virus and AIDS virus. Accordingly, it has been desired to develop antiviral agents having satisfactory effects against the viruses developing rapid variations.
For example, it is known that the two distinct proteins that are possessed by influenza virus, i.e., sialidase and hemagglutinin, have different roles. Sialidase is an enzyme which engages in the cleavage of sialic acid residues from sialyloligosaccharides on cellular surfaces when viruses germinate from cells. Recently, a sialic acid derivative having inhibitory activity against sialidase has been developed, and a clinical application as an anti-influenza viral agent is being studied.
Hemagglutinin, a trimer protein, has a key role in the introduction of a viral genome RNA into host cells by recognizing a sialyloligosaccharide, that has a specific binding mode upon infection to impart a host specificity to viruses, and by invading inside cells through endocytosis, and then changing its conformation and inducing the fusion of membranes. As to inhibitors of hemagglutinin, researches were conducted only from fundamental standpoints by using compounds comprised of a polyacrylamide bound by sialic acid (Mammen, M., et al., J. Med. Chem., 38, pp.4179-4190, 1995).
It is known that cholera toxin produced by Vibrio parahaemolyticus causes severe diarrhea. The toxin is composed of subunit A and subunit B. The subunit B, as a pentamer, acts as a receptor for a ligand, and has a role in introducing the subunit A as a toxic body, per se, into cytoplasm.
The object of the present invention is to provide a compound which exerts antiviral activity against variety of viruses and is useful as an active ingredient of a medicament. The inventors of the present invention conducted various studies to achieve the foregoing object, and as a result, found that a compound composed of a polymer containing a saccharide-conjugated sphingosine exhibited strong antiviral effect against viruses including influenza virus, for example. The present invention was achieved on the basis of these findings.
The present invention thus provides a polymer compound and a salt thereof which comprises a biodegradable polymer containing a glycosphingolipid. According to preferred embodiments of the present invention, the aforementioned polymer compound or a salt thereof wherein the glycosphingolipid is a lysoglycosphingolipid, more preferably lysoganglioside, and most preferably lysoganglioside GM3 is provided. According to further preferred embodiments, the aforementioned polymer compound or a salt thereof wherein the glycosphingolipid and the biodegradable polymer are bound by means of a spacer; and the aforementioned polymer compound or a salt thereof wherein the biodegradable polymer is a polyglutamic acid, more preferably a polyglutamic acid acetylated at N-terminal thereof are provided. Sodium salts may preferably be used as the salt of the compound.
According to the most preferred embodiment of the present invention, it is provided a polymer compound represented by the following formula (I) and a salt thereof: 
wherein R1 represents hydrogen atom or acetyl group; R2 represents hydrogen atom or a fluorescent functional group, and m and n independently represents an integer of from 10 to 40. According to a preferred embodiment of the aforementioned polymer compound and a salt thereof, there is provided the polymer compound and sodium salt thereof wherein R1 is acetyl group, R2 is a fluorescent functional group such as BODIPY group, m is an integer of from 15 to 25, n is an integer of from 20 to 40, and the amount of conjugated lysoganglioside GM3 is from 1 to 10 molar percent. Among them, the polymer compound and sodium salt thereof wherein m is 19, n is 27, and the amount of the conjugated lysoganglioside GM3 is 5 molar percent are most preferred.
According to another aspect of the present invention, there is provided a medicament comprising the aforementioned polymer compounds. According to preferred embodiment of the aforementioned invention, the medicament used as an antiviral agent and/or antidote is provided. According to further aspects of the present invention, there are provided a method for the treatment of an infectious disease caused by a virus or a microorganism, which comprises the step of administering a therapeutically effective amount of the aforementioned polymer compound to a patient; and lysoganglioside GM3 derivatives represented by the following formula (II): 
wherein R3 represents hydrogen atom or an amino protective group; and R4 represents hydrogen atom or a fluorescent functional group. The compound represented by the formula (II) is useful as a synthetic intermediate for the aforementioned polymer compound.
The polymer compounds of the present invention are characterized in that they comprise a biodegradable polymer containing a residue of a glycosphingolipid. As glycosphingolipids from which the residue of a glycosphingolipid is derived, any glycolipids may be used so long as they contain a long chain amino alcohol having 16 to 20 carbon atoms (sphingoid). Glycolipids containing a sphingosine may preferably be used, and most preferably, a naturally occurring sphingosine [D(+)-erythro-1,3-dihydroxy-2-amino-4-trans-octadecene] may be used. As the residue of the glycolipid, which corresponds to a partial structure of the glycosphingolipid, for example, a residue of a glycolipid formed by the conjugation of a sialic acid residue and a saccharide residue, and a residue of a glycolipid formed by the conjugation of a sialic acid residue and a nonreducing terminal of an oligosaccharide residue composed of two or more saccharide residues may preferably be used.
As the residues of glycosphingolipids, for example, lysoglycosphingolipid residues may preferably be used. Among them, a lysoganglioside residue (the term xe2x80x9clysogangliosidexe2x80x9d used herein means a ganglioside whose ceramide is deacylated, namely, a compound comprising a sialic acid-containing oligosaccharide chain bound to a sphingosine by means of a glycosidic linkage) is more preferred, and lysoganglioside GM3 (Lyso GM3) residue is most preferred.
Types of the biodegradable polymer are not particularly limited so long as they are readily degraded by enzymes existing in a living body, preferably in a human living body, and are substantially free from antigenicity and low toxic to a living body. Numbers of such biodegradable polymers are known, and an appropriate polymer is readily obtainable and used by one of those skilled in the art. For example, synthetic polymers as well as polypeptides, polynucleotides, polysaccharides and other may be used. When a serum half-life of the biodegradable polymer is too short, desired antiviral effects may sometimes be insufficiently achieved. On the other hand, if a serum half-life is too long, accumulation in a body may sometimes be a problem. Therefore, it is desired that a suitable type of biodegradable polymer may be chosen depending on the type of the glycosphingolipid, a desired biological action and other factors.
For example, polypeptides such as polyglutamic acid may preferably be used as the biodegradable polymer. When polyglutamic acids are used as the biodegradable polymer, for example, those having the degree of polymerization of about 100 to 1,000, preferably about 300 to 700, and most preferably about 500 to 600 can be used. In the polymer compounds represented by the general formula (I) mentioned above, it is preferred that m is an integer of from 15 to 25, n is an integer of from 20 to 40, and the amount of conjugated lysoganglioside GM3 is 1 to 10 molar percent.
In the polymer compounds of the present invention, the biodegradable polymer may be directly bound to the residue of the glycosphingolipid, or alternatively, they may be bound by means of a suitable spacer. In general, as to the linkage between the biodegradable polymer and the residue of the glycosphingolipid, a carboxyl group existing in the biodegradable polymer and an amino group existing in the sphingoid may preferably participate in the linkage of both units. When both units are directly bound to each other, it is preferred that an acid amide bond is formed between the carboxyl group and the amino group.
For example, when both units are bound to each other by means of a spacer, a carboxyl group and an amino group of a spacer, which consists of one amino acid residue, may bind to an amino group of the sphingoid and a carboxyl group of the biodegradable polymer, respectively, to form acid amide bonds. Alternatively, it is preferred that a C-terminal and a N-terminal of an oligopeptide spacer consisting of two or more amino acid residues may bind to an amino group of the sphingoid and a carboxyl group of the biodegradable polymer, respectively, to form acid amide bonds. However, It should be understood that the spacers and the linkages between the biodegradable polymer and the glycosphingolipid residue are not limited to those explained above, and they can be appropriately chosen by one of ordinary skilled in the art from various available spacers and bonding modes. For example, diamino compounds, acid anhydrides and other may be reacted to use as a spacer, or ester bond and other may be used as the linkage.
For example, the polymer compound of the present invention may have a fluorescent functional group so that intracellular transmigration can be visually traced after coupling with a virus. The fluorescent functional group may preferably be bound to, for example, a spacer moiety. Types of the fluorescent functional group are not particularly limited. For example, BODIPY group (4,4-difluoro-5,7-dimethyl-4-bora-3xcex1,4xcex1-diaza-s-indacene-3-propionic acid) may be used. When the fluorescent functional group is bound to a spacer, tri-functional amino, e.g., lysine, may preferably be used as a spacer which consists of one amino acid.
The polymer compound of the present invention may exist as the form of a salt such as sodium salt and potassium salt, and may also exist as a hydrate or a solvate. Hydroxyl groups, carboxyl groups, amino groups or other of the polymer compound of the present invention may be protected by using suitable protective groups. The protective groups are not particularly limited, and any types of protective groups available to those skilled in the art can be utilized. The polymer compound of the present invention may also exist as isomers in a pure form such as optically active compounds or diastereoisomers, or alternatively, exist as mixtures thereof (racemates and mixtures of diastereoisomers). Any of the substances mentioned above fall within the scope of the present invention.
Among preferred polymer compounds of the present invention, a particularly preferred compound (sodium salt) is shown below. However, the polymer compound of the present invention is not limited to the aforementioned particular compound. In the formula, m is 19, n is 27, and the amount of conjugated lysoganglioside GM3 is 5 molar percent. 
The polymer compound of the present invention, having a polar portion and a non-polar portion in the molecule, forms self micells in an aqueous solution, and behaves in the same manner as naturally occurring glycoproteins in a living body. Although it is not intended to be bound by any specific theory, the preferred polymer compound of the present invention shown as formula 5 is specifically recognized by a sialyloligosaccharide-recognizing receptor protein such as hemagglutinin of influenza virus and cholera toxin. After the conjugation with hemagglutinin, cholera toxin or other, the polymer compound changes its state from self-association to a linear chain structure, and strongly binds to side chains of hydrophobic amino acids of a protein or virus membranes by means of hydrophobic bond. Even if sialic acid is cleaved by sialidase deriving from influenza virus, the exposed galactose residues are caught by macrophages, and viruses, per se, are phagocytized by macrophages.
Accordingly, the polymer compound of the present invention is useful as an active ingredient of a medicament for preventive and/or therapeutic treatment of infectious diseases caused by viruses or microorganisms. In general, viruses have a feature of specifically recognizing a certain glycolipid or lipid, and therefore, a medicament which specifically acts on a specific virus or a microorganism can be produced by appropriately choosing a glycolipid portion of the glycosphingolipid residue of the polymer compound of the present invention. As an example, a polymer compound specifically acting on influenza virus is shown as formula 5. It is also possible to produce a medicament acting on plural viruses by choosing a glycolipid which is commonly recognized by several virus groups.
The medicament of the present invention may preferably be applicable to diseases such as, for example, viral hepatitis (type A, B, C, or E); influenza: virus pneumonitis; viral bronchitis; herpes infection, acute anterior poliomyelitis, AIDS, adult T cell leukemia, papilloma, measles, rubella, roseola infantum, erythema infectiosum, viral encephalitis, viral meningitis, cytomegalovirus infection, epidemic parotitis, chicken pox, hydrophobia, viral enteritis and the like. Among them, influenza is a particularly suitable target disease. However, diseases to which the medicament of the present invention is applicable are not limited to these viral diseases. The medicament of the present invention, in which the glycolipid portion of the polymer compound is appropriately chosen, may also be used for the treatment of infectious diseases caused by microorganisms such as bacteria.
The aforementioned polymer compound or a physiologically acceptable salt thereof may be used, per se, as the medicament of the present invention. However, it is generally preferred that a pharmaceutical composition for administration which comprises the above substance as an active ingredient is prepared by using additives for pharmaceutical preparations available to those skilled in the art. Although the route of administration of the medicament of the present invention is not particularly limited, it is preferred that the medicament is parenterally administered by using formulations such as injections, drip infusions, suppositories, inhalants, transmucosal preparations, transdermal preparations, nasal drops, ear drops and other. The medicament of the present invention may also be orally administered depending on a type of the biodegradable polymer. In that case, pharmaceutical preparations for oral administration such as tablets, capsules, powders, subtilized granules, granules, solutions, syrups and other can be used.
Examples of pharmacologically and pharmaceutically acceptable additives for pharmaceutical preparations include, for example, aqueous media such as distilled water for injection; solubilizers or solubilizing aids which can constitute injections dissolved before use; isotonic agents such as glucose; pH modifiers such as inorganic acids, organic acids, inorganic bases, and organic bases and the like. A dose of the medicament of the present invention should be suitably increased or decreased depending on various conditions such as a type of a disease, symptoms and the age of a patient, and a purpose of preventive or therapeutic treatment. The dose may be suitably chosen by those skilled in the art on the basis of these factors.
As an example of the method of preparation of the polymer compound, a method using lysoganglioside GM3 as a synthetic intermediate, represented by the general formula (II) wherein R3 is Fmoc group as an amino protective group and R4 is BODIPY group, will be described below in Examples. Accordingly, any desired polymer compounds falling within the general formula (I) may be readily prepared by referring to these methods, or by appropriately modifying or changing starting materials, reagents, reaction conditions and other. However, the methods of preparation of the polymer compound of the present invention are not limited to those described in the following examples.